Compositions, devices, and methods of mitigating lipoprotein interference in in vitro diagnostic assays for hydrophobic analytes

ABSTRACT

Methods of mitigating lipoprotein interference in in vitro diagnostic assays for target hydrophobic analytes are disclosed, as well as compositions, kits, and devices useful in said methods. A pretreatment reagent is utilized that includes at least one enzyme that digests lipoprotein.

CROSS REFERENCE TO RELATED APPLICATIONS/INCORPORATION BY REFERENCESTATEMENT

Not Applicable.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

Not Applicable.

BACKGROUND

In vitro diagnostic assays are used for the detection of hydrophobicanalytes in biological samples. However, lipoproteins (such as, but notlimited to, low density lipoprotein (LDL), very low density lipoprotein(VLDL), intermediate density lipoprotein (IDL), high density lipoprotein(HDL), and chylomicrons, etc. that are often quantitated by theircholesterol content) present in biological samples are known tointerfere with such assays, especially for small hydrophobic analytes(such as, but not limited to, hydrophobic drugs and hormones). Currentlythere are no methods to eliminate this interference, because thesehydrophobic haptens, when present in hydrophilic blood or serum, tend toinsert themselves into the hydrophobic core of lipoprotein particles. Assuch, a negative interference by lipoproteins (cholesterol) is oftenobserved because some of the analyte is tied up in the lipoproteinparticles and thus unavailable to the assay reagents.

Current attempts at overcoming cholesterol or lipoprotein interferenceinvolve extraction of the analyte using organic solvent. This approachbreaks down the lipoprotein so that the hydrophobic analyte becomesdissolved in the organic solvent. However, the extraction steps must beperformed manually, and thus are not amenable for mitigatinginterference in assays performed on fully automated analyzers.

In another approach, organic solvent or detergent is added to thereagent mixture to help solubilize the analyte and partially prevent itfrom entering into the hydrophobic core of lipoprotein particles.However, mitigation of lipoprotein interference by this approach islimited and cannot completely remove lipoprotein interference.

An ELISA assay was previously developed for the small, immunosuppressivedrug tacrolimus (also known as FK506) that utilizes proteinase to digestthe immunophilin FK506-binding protein (FKBP) and thus free up anytacrolimus bound by FKBP. However, the use of proteinase to digestprotein has limited effect on cholesterol/lipoprotein interference inthe assay.

Thus, new and improved methods of mitigating lipoprotein interference,especially for use with automated analyzers are needed. It is to suchmethods, as well as compositions, kits, and devices utilized in same,that the present disclosure is directed.

DETAILED DESCRIPTION

Before explaining at least one embodiment of the inventive concepts indetail by way of exemplary language and results, it is to be understoodthat the inventive concepts are not limited in their application to thedetails of construction and the arrangement of the components set forthin the following description. The inventive concepts are capable ofother embodiments or of being practiced or carried out in various ways.As such, the language used herein is intended to be given the broadestpossible scope and meaning, and the embodiments are meant to beexemplary—not exhaustive. Also, it is to be understood that thephraseology and terminology employed herein is for the purpose ofdescription only and should not be regarded as limiting.

Unless otherwise defined herein, scientific and technical terms used inconnection with the present disclosure shall have the meanings that arecommonly understood by those of ordinary skill in the art. Further,unless otherwise required by context, singular terms shall includepluralities and plural terms shall include the singular. The foregoingtechniques and procedures are generally performed according toconventional methods well known in the art and as described in variousgeneral and more specific references that are cited and discussedthroughout the present specification. The nomenclatures utilized inconnection with, and the laboratory procedures and techniques of,analytical chemistry, synthetic organic chemistry, and medicinal andpharmaceutical chemistry described herein are those well-known andcommonly used in the art. Standard techniques are used for chemicalsyntheses and chemical analyses.

All patents, published patent applications, and non-patent publicationsmentioned in the specification are indicative of the level of skill ofthose skilled in the art to which the present disclosure pertains. Allpatents, published patent applications, and non-patent publicationsreferenced in any portion of this application are herein expresslyincorporated by reference in their entirety to the same extent as ifeach individual patent or publication was specifically and individuallyindicated to be incorporated by reference.

All of the articles, compositions, kits, and/or methods disclosed hereincan be made and executed without undue experimentation in light of thepresent disclosure. While the articles, compositions, kits, and/ormethods have been described in terms of particular embodiments, it willbe apparent to those of skill in the art that variations may be appliedto the articles, compositions, kits, and/or methods and in the steps orin the sequence of steps of the methods described herein withoutdeparting from the concept, spirit, and scope of the present disclosure.All such similar substitutions and modifications apparent to thoseskilled in the art are deemed to be within the spirit, scope, andconcept of the inventive concepts as defined by the appended claims.

As utilized in accordance with the present disclosure, the followingterms, unless otherwise indicated, shall be understood to have thefollowing meanings:

The use of the term “a” or “an” when used in conjunction with the term“comprising” in the claims and/or the specification may mean “one,” butit is also consistent with the meaning of “one or more,” “at least one,”and “one or more than one.” As such, the terms “a,” “an,” and “the”include plural referents unless the context clearly indicates otherwise.Thus, for example, reference to “a compound” may refer to one or morecompounds, two or more compounds, three or more compounds, four or morecompounds, or greater numbers of compounds. The term “plurality” refersto “two or more.”

The use of the term “at least one” will be understood to include one aswell as any quantity more than one, including but not limited to, 2, 3,4, 5, 10, 15, 20, 30, 40, 50, 100, etc. The term “at least one” mayextend up to 100 or 1000 or more, depending on the term to which it isattached; in addition, the quantities of 100/1000 are not to beconsidered limiting, as higher limits may also produce satisfactoryresults. In addition, the use of the term “at least one of X, Y, and Z”will be understood to include X alone, Y alone, and Z alone, as well asany combination of X, Y, and Z. The use of ordinal number terminology(i.e., “first,” “second,” “third,” “fourth,” etc.) is solely for thepurpose of differentiating between two or more items and is not meant toimply any sequence or order or importance to one item over another orany order of addition, for example.

The use of the term “or” in the claims is used to mean an inclusive“and/or” unless explicitly indicated to refer to alternatives only orunless the alternatives are mutually exclusive. For example, a condition“A or B” is satisfied by any of the following: A is true (or present)and B is false (or not present), A is false (or not present) and B istrue (or present), and both A and B are true (or present).

As used herein, any reference to “one embodiment,” “an embodiment,”“some embodiments,” “one example,” “for example,” or “an example” meansthat a particular element, feature, structure, or characteristicdescribed in connection with the embodiment is included in at least oneembodiment. The appearance of the phrase “in some embodiments” or “oneexample” in various places in the specification is not necessarily allreferring to the same embodiment, for example. Further, all referencesto one or more embodiments or examples are to be construed asnon-limiting to the claims.

Throughout this application, the term “about” is used to indicate that avalue includes the inherent variation of error for acomposition/apparatus/device, the method being employed to determine thevalue, or the variation that exists among the study subjects. Forexample, but not by way of limitation, when the term “about” isutilized, the designated value may vary by plus or minus twenty percent,or fifteen percent, or twelve percent, or eleven percent, or tenpercent, or nine percent, or eight percent, or seven percent, or sixpercent, or five percent, or four percent, or three percent, or twopercent, or one percent from the specified value, as such variations areappropriate to perform the disclosed methods and as understood bypersons having ordinary skill in the art.

As used in this specification and claim(s), the words “comprising” (andany form of comprising, such as “comprise” and “comprises”), “having”(and any form of having, such as “have” and “has”), “including” (and anyform of including, such as “includes” and “include”), or “containing”(and any form of containing, such as “contains” and “contain”) areinclusive or open-ended and do not exclude additional, unrecitedelements or method steps.

The term “or combinations thereof” as used herein refers to allpermutations and combinations of the listed items preceding the term.For example, “A, B, C, or combinations thereof” is intended to includeat least one of: A, B, C, AB, AC, BC, or ABC, and if order is importantin a particular context, also BA, CA, CB, CBA, BCA, ACB, BAC, or CAB.Continuing with this example, expressly included are combinations thatcontain repeats of one or more item or term, such as BB, AAA, AAB, BBC,AAABCCCC, CBBAAA, CABABB, and so forth. The skilled artisan willunderstand that typically there is no limit on the number of items orterms in any combination, unless otherwise apparent from the context.

As used herein, the term “substantially” means that the subsequentlydescribed event or circumstance completely occurs or that thesubsequently described event or circumstance occurs to a great extent ordegree. For example, when associated with a particular event orcircumstance, the term “substantially” means that the subsequentlydescribed event or circumstance occurs at least 80% of the time, or atleast 85% of the time, or at least 90% of the time, or at least 95% ofthe time. The term “substantially adjacent” may mean that two items are100% adjacent to one another, or that the two items are within closeproximity to one another but not 100% adjacent to one another, or that aportion of one of the two items is not 100% adjacent to the other itembut is within close proximity to the other item.

As used herein, the phrases “associated with” and “coupled to” includeboth direct association/binding of two moieties to one another as wellas indirect association/binding of two moieties to one another.Non-limiting examples of associations/couplings include covalent bindingof one moiety to another moiety either by a direct bond or through aspacer group, non-covalent binding of one moiety to another moietyeither directly or by means of specific binding pair members bound tothe moieties, incorporation of one moiety into another moiety such as bydissolving one moiety in another moiety, and coating one moiety onanother moiety, for example.

The term “sample” as used herein will be understood to include any typeof biological sample that may be utilized in accordance with the presentdisclosure. Examples of fluidic biological samples that may be utilizedinclude, but are not limited to, whole blood or any portion thereof(including, but not limited to, plasma or serum), whole or lysed bloodcells (including, but not limited to, whole or lysed red blood cells),urine, saliva, sputum, cerebrospinal fluid (CSF), skin, intestinalfluid, intraperitoneal fluid, cystic fluid, sweat, interstitial fluid,extracellular fluid, tears, mucus, bladder wash, semen, fecal, pleuralfluid, nasopharyngeal fluid, combinations thereof, and the like.

The term “hapten” as used herein refers to a small proteinaceous ornon-protein antigenic determinant (or “epitope”) which is capable ofbeing recognized in an in vitro diagnostic assay (which may involve atarget analyte-specific binding partner, such as (but not limited to) anantibody).

The term “analyte” refers to a molecule that is capable of beingrecognized in an in vitro diagnostic assay (which may involve a targetanalyte-specific binding partner, such as (but not limited to) anantibody). When the assay is an immunoassay, the analyte comprises atleast one antigenic determinant or “epitope,” which is the region of theanalyte which binds to the target analyte-specific binding partner(i.e., antibody). When the analyte is a hapten, the entire haptenmolecule typically forms the epitope.

The term “target analyte-specific binding partner” as used herein willbe understood to refer to any molecule capable of specificallyassociating with the target analyte. For example but not by way oflimitation, the binding partner may be an antibody, a receptor, aligand, aptamers, molecular imprinted polymers (i.e., inorganicmatrices), combinations or derivatives thereof, as well as any othermolecules capable of specific binding to the target analyte.

The term “antibody” is used herein in the broadest sense and refers to,for example, intact monoclonal antibodies and polyclonal antibodies,multi-specific antibodies (e.g., bispecific antibodies), antibodyfragments and conjugates thereof that exhibit the desired biologicalactivity of analyte binding (such as, but not limited to, Fab, Fab′,F(ab′)2, Fv, scFv, Fd, diabodies, single-chain antibodies, and otherantibody fragments and conjugates thereof that retain at least a portionof the variable region of an intact antibody), antibody substituteproteins or peptides (i.e., engineered binding proteins/peptides), andcombinations or derivatives thereof. The antibody can be of any type orclass (e.g., IgG, IgE, IgM, IgD, and IgA) or sub-class (e.g., IgG1,IgG2, IgG3, IgG4, IgA1, and IgA2).

The term “microfluidics device” as used herein includes any device(s)capable of performing at least one diagnostic assay as described herein.The microfluidics device will typically be inserted into a system thatautomates the performance of the diagnostic assay(s). In onenon-limiting embodiment, the microfluidics device is constructed for usein automated diagnostic assays conducted by, for example but not by wayof limitation, one of the DIMENSION® integrated chemistry systemscommercially available from Siemens Healthcare Diagnostics, Inc.(Newark, Del.). However, it will be understood that the microfluidicsdevice can be any commercially available product described or otherwisecontemplated herein that is capable of performing one or more diagnosticassays in accordance with the present disclosure. In addition, themicrofluidics device may include multiple compartments that integratemultiple assays onboard a single microfluidics device used with aclinical chemistry system, so that a single amount of a biologicalsample is inserted into the microfluidics device and then delivered tomultiple assay compartments.

Turning now to the inventive concepts, certain non-limiting embodimentsof the present disclosure relate generally to compositions, kits,devices, and methods for improving the performance and reliability of invitro diagnostic assays for hydrophobic analytes. In particular, certainembodiments of the present disclosure are related to compositions, kits,devices, and methods for mitigating lipoprotein interference in in vitrodiagnostic assays for hydrophobic analytes.

Certain non-limiting embodiments of the present disclosure are directedto methods for detecting the presence and/or concentration of a targethydrophobic analyte in a biological sample. In certain particular (butnon-limiting) embodiments, the methods may be further defined as methodsof minimizing lipoprotein interference in in vitro diagnostic assays forhydrophobic analytes.

The methods include combining, either simultaneously or wholly orpartially sequentially: (1) a sample suspected of containing the targethydrophobic analyte; (2) a pretreatment reagent comprising at least oneenzyme that digests lipoprotein (such as, but not limited to, lipaseand/or at least one other digestive enzyme (such as, but not limited to,stomach enzymes such as (but not limited to) pepsin and pancreaticenzymes such as (but not limited to) amylase and protease); and (3) atleast one assay reagent capable of detecting a hydrophobic analyte. Themethod further includes performing one or more detection assays based on(3) and determining a concentration of target hydrophobic analytepresent in the sample.

When (1) and (2) are incubated together, the enzyme present in thepretreatment reagent digests lipid and protein in the lipoprotein. Thiscauses release of target analyte from the hydrophobic core of alipoprotein particle so that the target analyte becomes accessible tothe assay reagent(s) (i.e., such as, but not limited to, antibodies thatspecifically recognize the target analyte). As such, the target analyteis detected at an increased efficiency compared to assays in which thepretreatment reagent is not utilized, thereby providing a more robustassay for the target analyte.

One or more of the method steps can be performed manually;alternatively, the method steps may be fully automated on a clinicalchemistry analyzer system, as described herein.

Any biological sample known in the art for use with in vitro diagnosticassays as described herein may be utilized in accordance with thepresent disclosure. Examples of biological samples that may be utilizedinclude, but are not limited to, urine, whole blood or any portionthereof (including, but not limited to, plasma or serum), whole (i.e.,substantially unlysed) or lysed blood cells (including, but not limitedto, whole or lysed red blood cells), saliva, sputum, cerebrospinal fluid(CSF), intestinal fluid, intraperitoneal fluid, cystic fluid, sweat,interstitial fluid, tears, mucus, bladder wash, semen, combinations, andthe like. Any hydrophobic analytes capable of detection via the assaymethods disclosed or otherwise contemplated herein may be detected viathe methods of the present disclosure. Examples of target analytesinclude small hydrophobic molecules such as (but not limited to) vitaminD, tacrolimus, sirolimus, everolimus, estrogen, estrone, estradiol,estriol, alfatradiol (estradiol, estrone, and estriol), cyclosporine,ethinylestradiol, esterified estrogens, moxestrol, qunestrol,progestins, progesterone, androgens such as testosterone,dihydroteestosterione (DHT), dehydroepiandrosterone (DHEA) and DHEAsulfate (DEHE-S), androstenendione aldosterone, other steroid horomones,cortisol, catecholamine, 25-hydroxy Vitamin D2 (25-OH Vitamin D2),25-hydroxy Vitamin D3 (25-OH Vitamin D3), 1,25-dihydroxy Vitamin D2(1,25-OH Vitamin D2), and 1,25-dihydroxy Vitamin D3 (1,25-OH VitaminD3), and the like.

The pretreatment reagent comprises at least one enzyme that digestslipids on lipoprotein into more water soluble and smaller compounds;said enzyme may be lipase and/or at least one other digestive enzyme(such as, but not limited to, stomach enzymes such as (but not limitedto) pepsin and pancreatic enzymes such as (but not limited to) amylaseand protease). In addition, the pretreatment reagent may comprise one ormore additional substances that function to enhance enzymatic activity.Non-limiting examples of additional substances that may be present inthe pretreatment reagent include at least one cofactor for the enzyme(such as, but not limited to, bile acid and/or a salt thereof), at leastone surfactant (such as, but not limited to, PLURONIC® block copolymer(BASF Corporation, Ludwigshafen, Germany), or at least one protease, aswell as any combination thereof.

The enzyme that digests lipoprotein must be provided at a sufficientconcentration so as to cause the hydrophobic core of a lipoproteinparticle to release the target analyte so that it becomes accessible tothe assay reagent(s), thereby allowing for detection of the targetanalyte at increased efficiency. The concentration of enzyme will bedependent upon the amount of analyte present and the time of thepretreatment reaction.

The enzyme(s) that digests lipoprotein is present in the pretreatmentreagent may be present at any concentration that allows the enzyme tofunction as described herein. Examples of particular concentrationsinclude, but are not limited to: about 0.01 mg/mL, about 0.05 mg/mL,about 0.1 mg/mL, about 0.2 mg/mL, about 0.3 mg/mL, about 0.4 mg/mL,about 0.5 mg/mL, about 0.6 mg/mL, about 0.7 mg/mL, about 0.8 mg/mL,about 0.9 mg/mL, about 1 mg/mL, about 1.5 mg/mL, about 2 mg/mL, about2.5 mg/mL, about 3 mg/mL, about 3.5 mg/mL, about 4 mg/mL, about 4.5mg/mL, about 5 mg/mL, about 5.5 mg/mL, about 6 mg/mL, about 6.5 mg/mL,about 7 mg/mL, about 7.5 mg/mL, about 8 mg/mL, about 8.5 mg/mL, about 9mg/mL, about 9.5 mg/mL, about 10 mg/mL, about 11 mg/mL, about 12 mg/mL,about 13 mg/mL, about 14 mg/mL, about 15 mg/mL, about 16 mg/mL, about 17mg/mL, about 18 mg/mL, about 19 mg/mL, about 20 mg/mL, about 21 mg/mL,about 22 mg/mL, about 23 mg/mL, about 24 mg/mL, about 25 mg/mL, about 26mg/mL, about 27 mg/mL, about 28 mg/mL, about 29 mg/mL, about 30 mg/mL,about 31 mg/mL, about 32 mg/mL, about 33 mg/mL, about 34 mg/mL, about 35mg/mL, about 36 mg/mL, about 37 mg/mL, about 38 mg/mL, about 39 mg/mL,about 40 mg/mL, about 41 mg/mL, about 42 mg/mL, about 43 mg/mL, about 44mg/mL, about 45 mg/mL, about 46 mg/mL, about 47 mg/mL, about 48 mg/mL,about 49 mg/mL, about 50 mg/mL, about 55 mg/mL, about 60 mg/mL, about 65mg/mL, about 70 mg/mL, about 75 mg/mL, about 80 mg/mL, about 85 mg/mL,about 90 mg/mL, about 95 mg/mL, about 100 mg/mL, or higher. In aparticular (but non-limiting) embodiment, the concentration of theenzyme in the pretreatment reagent is defined as being within a range ofany two of the values shown herein above, such as (but not limited to) arange of from about 0.01 mg/mL to about 100 mg/mL, a range of from about0.05 mg/mL to about 50 mg/mL, a range of from about 0.1 mg/mL to about20 mg/mL, a range of from about 0.5 mg/mL to about 10 mg/mL, and thelike. However, it is to be understood that the specific concentrationranges listed above are for illustration purposes only and are not to beconsidered limiting; any concentration range having a lower limit of oneof the values listed above and an upper limit of another of the valueslisted above explicitly falls within the scope of the presentdisclosure.

Similarly, each of the one or more additional substances of thepretreatment reagent may be present at any concentration that allows thesubstance(s) to function to enhance enzymatic activity. For example,each of the additional substances (such as, but not limited to, theenzyme cofactor like bile acid and/or a salt thereof, the surfactantlike PLURONIC® block copolymer, or a protease), may be provided at aconcentration selected from the following non-limiting examples ofconcentrations: about 0.001%, about 0.005%, about 0.01%, about 0.02%,about 0.03%, about 0.04%, about 0.05%, about 0.06%, about 0.07%, about0.08%, about 0.09%, about 0.1%, about 0.15%, about 0.2%, about 0.25%,about 0.3%, about 0.35%, about 0.4%, about 0.45%, about 0.5%, about0.55%, about 0.6%, about 0.65%, about 0.7%, about 0.75%, about 0.8%,about 0.85%, about 0.9%, about 0.95%, about 1%, about 1.1%, about 1.2%,about 1.3%, about 1.4%, about 1.5%, about 1.6%, about 1.7%, about 1.8%,about 1.9%, about 2%, about 2.1%, about 2.2%, about 2.3%, about 2.4%,about 2.5%, about 2.6%, about 2.7%, about 2.8%, about 2.9%, about 3%,about 3.25%, about 3.5%, about 3.75%, about 4%, about 5%, about 6%,about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%,about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, andhigher. In a particular (but non-limiting) embodiment, the concentrationof each additional substance present in the pretreatment reagent isdefined as being within a range of any two of the values shown hereinabove, such as (but not limited to) a range of from about 0.001% toabout 25%, a range of from about 0.005% to about 15%, a range of fromabout 0.01% to about 5%, and the like. However, it is to be understoodthat the specific concentration ranges listed above are for illustrationpurposes only and are not to be considered limiting; any concentrationrange having a lower limit of one of the values listed above and anupper limit of another of the values listed above explicitly fallswithin the scope of the present disclosure.

In certain non-limiting embodiments, the at least one assay reagentcapable of detecting a hydrophobic analyte is an immunoassay reagent(i.e., a target analyte-specific binding partner (such as, but notlimited to, an antibody). The at least one target analyte-specificbinding partner is then allowed to bind to the target analyte or the atleast one immunoassay reagent.

The pretreatment and assay/detection steps may be performed in the samecompartment of a microfluidics device. Alternatively, the pretreatmentstep may be performed in a first compartment, and then the pretreatedsample transferred to a second compartment for performing the detectionstep.

The biological sample may be added to the pretreatment step in itsnative form, or the biological sample may be lysed prior to thepretreatment step. Thus, in a particular (but non-limiting) embodiment,the methods described or otherwise contemplated herein may furtherinclude the steps of lysing a biological sample in a first compartmentand then transferring the lysed biological sample to a secondcompartment utilized in the pretreatment step.

Any of the method steps described herein may be performed, for examplebut not by way of limitation, by a user. However, as used herein, theterm “user” is not limited to use by a human being; rather, the term“user” may comprise (for example, but not by way of limitation) acomputer, a server, a website, a processor, a network interface, ahuman, a user terminal, a virtual computer, combinations thereof, andthe like.

Certain non-limiting embodiments of the present disclosure are directedto reagent kits useful for conveniently performing the diagnostic assaymethods described herein above. The reagent kit includes one or more ofany of the pretreatment reagents described or otherwise contemplatedherein in combination with one or more of any of the assay reagentscapable of detecting a hydrophobic analyte as described or otherwisecontemplated herein.

Certain other non-limiting embodiments of the present disclosure aredirected to an assay device (such as, but not limited to, amicrofluidics device) which contains at least one of any of thepretreatment reagents described or otherwise contemplated herein and atleast one of any of the assay reagents described or otherwisecontemplated herein, and wherein the assay device is for use in any ofthe in vitro diagnostic assay methods described herein above.

For example, a microfluidics device may include at least one compartmentcapable of receiving a sample suspected of containing a targethydrophobic analyte, wherein the at least one compartment includes atleast one of the pretreatment reagents as described in detail hereinabove. The compartment may further contain one or more of any of theassay reagents described or otherwise contemplated herein.Alternatively, the microfluidics device may be provided with at least asecond compartment that is capable of being in fluidic communicationwith the first compartment containing the pretreatment reagent, and theassay reagent(s) may be included in the second compartment. The secondcompartment may be a read chamber where the detection assay fordetermining a concentration of target hydrophobic analyte present in thesample is performed; alternatively, the microfluidics device mayadditionally include a read chamber capable of being in fluidiccommunication with the second compartment.

In addition, the reagent kits and/or microfluidics devices of thepresent disclosure may further contain other component(s) and/orreagent(s) for conducting any of the particular diagnostic assaysdescribed or otherwise contemplated herein. The nature of theseadditional component(s)/reagent(s) will depend upon the particular assayformat, and identification thereof is well within the skill of one ofordinary skill in the art. Examples of additional reagents/componentsthat may be present in the reagent kits and/or microfluidics devices ofthe present disclosure include, but are not limited to, diluents, lysingagents (for lysing red blood cells), wash solutions (such as but notlimited to, isotonic solutions), positive controls, negative controls,quality controls, and/or actuators, as well as any combination thereof.

The relative amounts of the various components/reagents in the kitsand/or microfluidics devices can vary widely to provide forconcentrations of the components/reagents that substantially optimizethe reactions that need to occur during the assay methods and further tooptimize substantially the sensitivity of an assay.

The reagent kits of the present disclosure may further include a set ofwritten instructions explaining how to use the kit. A kit of this naturecan be used with any of the microfluidics devices and/or in any of themethods described or otherwise contemplated herein.

The microfluidics device may have one or more manual functionsassociated therewith (i.e., wherein pipetting is required for additionof one or more reagents and/or movement of a mixture between twocompartments); alternatively, the microfluidics device may be a fullyautomatic, closed system in which the necessary reagents/components aredisposed in various compartments during construction of the device(wherein the various compartments are in continuous fluidiccommunication (or are capable of being in continuous fluidiccommunication)), and thus no manual manipulation of the sample and/orreagent(s) is required for performance of the assay after the sample isadded to the microfluidics device.

As described herein above, the microfluidics device comprises one ormore compartments containing the components/reagents described hereinabove. However, it will be understood that the microfluidics device maybe provided with any number of compartments, any arrangement ofcompartments, and any distribution of the components/reagentstherebetween, so long as the device is able to function in accordancewith the present disclosure. When provided with multiple compartments,the compartments may be completely separated from one another, or one ormore compartments may be capable of being in fluidic communication withone another. Various structures of microfluidics devices that arecapable of use in accordance with the present disclosure are well knownin the art, and therefore no further description thereof is deemednecessary.

In certain embodiments, the microfluidics device includes at least firstand second compartments. The first compartment is capable of receiving abiological sample and, if desired (but not by way of limitation), mayinclude a mechanism for lysing red blood cells or otherwise preparingthe sample for pretreatment and assay. Said separation mechanisms arewell known in the art of microfluidics devices, and therefore no furtherdescription thereof is deemed necessary. The second compartment iscapable of being in fluidic communication with the first compartment andincludes the at least one pretreatment reagent. The second compartmentmay further include the at least one assay reagent; alternatively, themicrofluidics device may include a third compartment for storage of theat least one assay reagent, and wherein the at least one assay reagentcan be transferred from the third compartment into the secondcompartment following incubation of the sample with the pretreatmentreagent in a pretreatment step.

The microfluidics device may also include a read chamber (such as, butnot limited to, an optical read chamber) that is capable of beinginterrogated by a clinical chemistry system (such as, but not limitedto, optically interrogated by a spectrometer). The read chamber may beassociated with any of the compartments described herein above, or theread chamber may be associated with a separate compartment from thosedescribed herein above.

The inlet channel and a compartment, as well as two compartments, may bedescribed as being “capable of being in fluidic communication” with oneanother; this phrase indicates that the compartment(s) may still besealed, but the two compartments are capable of having fluid flowtherebetween upon puncture of a seal formed therein or therebetween.

The kits/microfluidics devices of the present disclosure may be providedwith any other desired features known in the art or otherwisecontemplated herein. For example, but not by way of limitation, thekits/microfluidics devices of the present disclosure may further includeone or more additional compartments containing other solutions, such asbut not limited to, lysing agents (for lysing red blood cells),diluents, wash solutions, labeling agents, interference solutions,positive controls, negative controls, quality controls, and/oractuators, as well as any combination thereof.

Example

An Example is provided hereinbelow. However, the present disclosure isto be understood to not be limited in its application to the specificexperimentation, results, and laboratory procedures disclosed herein.Rather, this Example is simply provided as one of various embodimentsand is meant to be exemplary, not exhaustive.

In this Example, the effects of the use of pretreatment reagents oncholesterol interference in an everolimus (EVRO) assay on a DIMENSION®integrated chemistry system (Siemens Healthcare Diagnostics, Inc.,Newark, Del.) were examined. Varying amounts of lipase, bile salt (as acofactor for lipase), and PLURONIC® block copolymer (BASF Corporation,Ludwigshafen, Germany) were spiked in pretreatment reagents that wereincubated with samples containing known amounts of cholesterol prior toperforming an everolimus (EVRO) assay of the treated sample on aDIMENSION® integrated chemistry system (Siemens Healthcare Diagnostics,Inc., Newark, Del.).

As shown in Table 1, in the absence of a pretreatment step, the amountof cholesterol interference observed in the EVRO assay was −7.6%, −12%,and −19% for cholesterol concentrations of 300 mg/mL, 350 mg/mL, and 400mg/mL, respectively. The addition of a pretreatment step with a reagentcomprising lipase, its cofactor bile acid/salt (for enzymatic activityenhancement), and PLURONIC® as surfactant decreased the amount ofcholesterol interference to 1% (from −7.6%), −0.4% (from −12%), and −3%(from −19%) at cholesterol concentrations of 300 mg/mL, 350 mg/mL, and400 mg/mL, respectively. As such, cholesterol interference wassubstantially eliminated at 400 mg/mL cholesterol using the compositionsand methods of the present disclosure.

TABLE 1 Effect of Lipase on Cholesterol Interference Mitigation Lipasemg/mL 0 5 5 3 5 % Bile salt    0%  0.1%  0.1%  0.1% 0.05% % Pluronic  0.9%  0.9%    0%   0%   0% 300 mg/mL Cholesterol  −7.6%  1.0% −0.6% 0.5% −1.6% 350 mg/mL Cholesterol −12.0% −0.4%  0.6% −6.9% −4.3% 400mg/mL Cholesterol −19.0% −3.0% −5.4% −5.6% −6.9%

Thus, in accordance with the present disclosure, there have beenprovided compositions, kits, and devices, as well as methods ofproducing and using same, which fully satisfy the objectives andadvantages set forth hereinabove. Although the present disclosure hasbeen described in conjunction with the specific drawings,experimentation, results, and language set forth hereinabove, it isevident that many alternatives, modifications, and variations will beapparent to those skilled in the art. Accordingly, it is intended toembrace all such alternatives, modifications, and variations that fallwithin the spirit and broad scope of the presently disclosed inventiveconcepts.

What is claimed is:
 1. A microfluidics device for use in a clinicalchemistry instrument, the microfluidics device comprising: at least onecompartment capable of receiving a sample suspected of containing atarget hydrophobic analyte, wherein the at least one compartmentcomprises: a pretreatment reagent comprising at least one enzyme thatdigests lipoprotein; and at least one assay reagent capable of detectinga hydrophobic analyte for determining a concentration of targethydrophobic analyte present in the sample.
 2. The microfluidics deviceof claim 1, wherein the microfluidics device is further defined ascomprising at least two compartments, wherein the pretreatment reagentis disposed in a first compartment that is capable of receiving thesample, and the at least one assay reagent is disposed in a secondcompartment that is capable of being in fluidic communication with thefirst compartment, and wherein the first compartment or the secondcompartment is a read chamber where the detection assay for determininga concentration of target hydrophobic analyte present in the sample isperformed.
 3. The microfluidics device of claim 1, wherein the at leastone enzyme that digests lipoprotein comprises lipase and/or at least onedigestive enzyme.
 4. The microfluidics device of claim 1, wherein theenzyme is present in the pretreatment reagent at a concentration in arange of from about 0.1 mg/mL to about 20 mg/mL.
 5. The microfluidicsdevice of claim 1, wherein the pretreatment reagent further comprises atleast one additional substance selected from the group consisting of acofactor for the enzyme, a surfactant, a protease, and combinationsthereof.
 6. The microfluidics device of claim 5, wherein the at leastone enzyme that digests lipoprotein is lipase, and wherein thepretreatment reagent comprises bile acid and/or a salt thereof as acofactor for lipase.
 7. The microfluidics device of claim 1, wherein thetarget hydrophobic analyte is selected from the group consisting ofvitamin D, tacrolimus, sirolimus, everolimus, estrogen, estrone,estradiol, estriol, alfatradiol, cyclosporine, ethinylestradiol,esterified estrogens, moxestrol, qunestrol, progestins, progesterone,androgens, testosterone, dihydroteestosterione (DHT),dehydroepiandrosterone (DHEA), DHEA sulfate, androstenendionealdosterone, cortisol, catecholamine, 25-hydroxy Vitamin D2, 25-hydroxyVitamin D3, 1,25-dihydroxy Vitamin D2, and 1,25-dihydroxy Vitamin D3. 8.The microfluidics device of claim 1, wherein the at least one assayreagent is further defined as an immunoassay reagent. 9.-22. (canceled)